Method of operating a scanning confocal imaging system

Abstract

A confocal scanning microscope operates to produce a small (preferably diffraction limited) spot on a sample, scan the spot over the sample in a raster pattern, and form an electrical signal corresponding to the intensity of light emanating from the region of the spot. The electrical signal is communicated to a computer which produces a visual display on a monitor. The optical train between the source (or detector) and the sample comprises first and second scanning elements, each operable to scan the beam in a different direction, an afocal assembly located between the two scanning elements so as to transfer the beam from the first scanning element to the second scanning element, and standard microscope components including an objective. The light emanating from the sample encounters the objective, the scanning elements, and the afocal assembly prior to reaching the detector. An aperture is disposed in front of the detector and blocks any light that emanated from points spatially displaced from the beam spot. The use of an afocal assembly in the optical train thus allows the elimination of pinhole spatial filters from the design.

I claim: 
     
       1. In the operation of a scanning confocal microscope, a method comprising the steps of: forming a beam spot;   collecting light emanating from the region of the beam spot;   directing the light so collected along a path;   collimating the light along the path, the light so collimated being generally characterized by a beam diameter;   passing the light through an aperture generally commensurate in size with the beam diameter, the path length to the aperture being relatively long compared to the aperture, the path being further devoid of pinhole spatial filters;   detecting the light passing through the aperture; and   said collecting, directing, collimating, and passing steps being performed to prevent light that emanates from points away from the beam spot location from passing through the aperture and being detected by said detecting step.